DESCRIPTION (the applicant's description verbatim): Mitochondrial Ca2+ transport plays a central role in regulating cellular Ca2+ homeostasis, energy metabolism, apoptosis, and necrosis. The long-term objective of our research is to elucidate an integrative mechanism by which mitochondria regulate intracellular Ca2+ signaling under both physiological and pathological conditions. Our immediate efforts, described in this proposal, are focused upon the characterization of mitochondrial Ca2+ influx and efflux mechanisms in cardiac muscle cells and determining how these mechanisms regulate excitation-contraction coupling. Our working hypothesis is that cardiac mitochondria contain a Ca2+-activated, ryanodine-sensitive and a Ca2+-activated, cyclosporin-sensitive Ca2+ permeable channel that are responsible for the fast uptake of Ca2+ into and fast release of Ca2+ out of mitochondria, respectively. These dynamic Ca2+ transport systems participate actively in regulating the cardiac excitation-contraction coupling process, due to their structural proximity to the junctions between sarcoplasmic reticulum and L-type Ca2+ channels. The specific aims of this proposal are (1) to characterize the mitochondrial Ca2+ influx mechanisms that are responsible for the fast uptake of Ca2+ into mitochondria. (2) To determine the frequency-dependence of mitochondrial Ca2+ uptake from the cytosol and its role in modulating recovery of L-type Ca2+ channel from inactivation. (3) To characterize the mitochondrial Ca2+ efflux mechanisms that are responsible for the fast pumping of Ca2+ out of mitochondria. We will use a multidisciplinary approach, encompassing single cell fluorescence confocal microscopy to measure cytosolic and mitochondrial Ca2+ concentrations, patch clamp to record L-type Ca2+ currents, and electron microscopy to determine the distance between mitochondria and individual sarcoplasmic reticulum Ca2+ release channels. The results from the present proposal will provide essential information regarding the importance of mitochondrial Ca2+ transport during normal cardiac function. This information is critical for our understanding of mitochondria-related cardiovascular diseases such as ischemic heart disease, cardiac arrhythmia, cardiomyopathy, and heart failure.